Muscle wasting remains a cause of morbidity and mortality after trauma, burns and infection. Although the mechanisms for the atrophic response are multifactorial and poorly defined, our work documents the causal relationship between the up regulation of inflammatory modulators, particularly TNF" and NO, and the decreased translational control of muscle protein synthesis. The sepsis-induced defect is manifested not only under basal conditions but also as a reduced responsiveness to leucine which acts as an anabolic nutrient signal. Our data suggest these sepsis-induced changes are mediated by inhibition of mTOR kinase signaling in skeletal muscle. Our long-term goal is to elucidate the cellular and molecular mechanisms by which sepsis impairs muscle protein balance leading to the development of muscle atrophy. To address the questions implicit in this goal our proposed research has the following specific aims: (1) Elucidate the mechanism by which sepsis impairs mTOR kinase activity and cap-dependent translation initiation in muscle under basal conditions and in response to leucine by determining whether sepsis alters the interaction of 4E-BP1 and S6K1 with the scaffold protein raptor, the binding activity of the eIF3 scaffolding complex, and/or the binding of PDCD4 to eIF4A and thereby altering helicase activity. (2) Delineate the mechanism by which sepsis impairs the topology of protein synthesis by altering the intracellular localization of components of the translational apparatus. Specifically, we will assess the role of Vps34, a nutrient-regulated lipid kinase, and Rab5 in regulating intracellular mTOR localization. (3) Determine whether sepsis decreases mTOR kinase activity by altering the phosphorylation and/or binding of the Akt substrate PRAS40. (4) Determine the mechanism by which sepsis-induced changes in microRNAs, particularly miR133, decrease global translation initiation. (5) Elucidate the relative contribution of systemic versus locally produced TNF" and NO in disrupting mTOR signaling in muscle. This final aim directly addresses the importance of autocrine/paracrine production of inflammatory mediators in sepsis-induced muscle wasting by using primary cultures of myocytes isolated from TNFR1 null mice or in complementary in vivo studies where shRNA specific for TNF" is electroporated locally into muscle. Collectively, these studies are unique as they integrate in vitro and in vivo approaches permitting detailed effector mechanisms to be defined and the physiological relevance of the observations to be confirmed. These studies will provide new insights into this critical regulatory system and the integrative control mechanisms exerted by a key nutrient signal. They will lead not only to a thorough understanding of sepsis- induced myopathy but also of other health-related conditions characterized by muscle wasting and nutrient resistance. The proposed studies have a translational underpinning in that they will stimulate the development of novel clinical interventions designed to abrogate the long-term consequences of sepsis- and trauma-induced myopathy which impedes recovery and rehabilitation. PUBLIC HEALTH RELEVANCE: The loss of muscle in patients after infection or traumatic injury delays recovery and may decrease survival. The reason why this wasting occurs is not fully understood, but it cannot be fully corrected by providing adequate nutrition. Our research will determine the mechanism for this muscle atrophy which will ultimately lead to development of novel therapeutic treatment and improve patient outcome.